High Resolution Gold Labels for EM

EM 高分辨率金标签

• 批准号: 7281220
• 负责人: James F. Hainfeld
• 金额: $ 45.97万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7281220
• 关键词: Address; Affect; Affinity; Antibodies; Arsenic; Binding; Biological; Biological Process; Charge; Chimeric Proteins; Chromatin Remodeling Factor; Complex; Couples; Cryoelectron Microscopy; Cysteine; DNA Repair; Data; Development; Dose; Electron Microscopy; Electrons; Engineering; G22P1 gene; Gold; Green Fluorescent Proteins; Human; Ice; Image; Label; Ligase; Link; Location; Methods; Molecular Biology; Muscle Rigidity; Negative Staining; Noise; Numbers; Positioning Attribute; Protein Engineering; Proteins; Repair Complex; Resolution; Signal Transduction; Site; Specificity; Structure; Technology; Testing; Uncertainty; Work; XRCC5 gene; base; density; image processing; improved; monomaleimido gold; new technology; particle; reconstruction; stem

DESCRIPTION (provided by applicant): Heavy atom labels can augment electron microscopy of biological structures by: a) revealing the site on a specific structure within a complex, and b) facilitating location and alignment of an overall complex. A useful EM label has two components: 1) a dense object which gives a high signal-to-noise ratio under the circumstances of observation, and 2) a linker which confers specificity, rigidity, and high occupancy without affecting biological function. Previously we have optimized labels for STEM. In this proposal we will better adapt the technology to cryoEM and modern molecular biology. New technology developed for protein engineering provides a variety of tags which can be inserted into the structure genetically and used for purification. These include: His-tags, Flag-tags, GST-tags, and strept-tags. Some of these same tags appear ideally suited as linkers for rigid, high-affinity attachment of density labels. Therefore, we propose to develop a labeling toolkit for cryoEM based on these new capabilities. In particular we will primarily evaluate five tags that bind to specialized gold clusters: 1) Ni-NTA-gold binding to 6x-His tags, 2) negatively charged gold to 6x Arg tags, 3) monomaleimido gold linked to cysteine, 4) arsenic-gold to tetra-Cys tags, and 5) gold targeted to a gold-binding sequence. These labels will then be used to determine subunit positions in several important protein complexes: The DNA repair complexes consisting of subcomplexes: human Ku70/Ku80/DNA-PK complex, human ligase IV/Xrcc4 complex, human RAD50/Mre11/Nbs1 complex, and the human chromatin remodeling complex ACF. The gold labels will additionally be used to better align the complexes to improve reconstructions from conical tilt pairs using cryoEM.

描述（由申请人提供）：重原子标记可以通过以下方式增强生物结构的电子显微镜检查：a）揭示复合物内特定结构上的位点，以及b）促进整个复合物的定位和对准。 有用的 EM 标签有两个组成部分：1) 在观察情况下提供高信噪比的致密物体，2) 赋予特异性、刚性和高占用率而不影响生物功能的连接体。之前我们已经优化了 STEM 的标签。在本提案中，我们将更好地将该技术应用于冷冻电镜和现代分子生物学。 为蛋白质工程开发的新技术提供了多种标签，可以通过基因方式将其插入结构中并用于纯化。这些包括：His 标签、Flag 标签、GST 标签和 strept 标签。其中一些相同的标签似乎非常适合作为密度标签的刚性、高亲和力附着的连接子。 因此，我们建议开发一个基于这些新功能的冷冻电镜标记工具包。特别是，我们将主要评估与特殊金簇结合的五个标签：1) Ni-NTA-金与 6x-His 标签结合，2) 带负电荷的金与 6x Arg 标签结合，3) 单马来酰亚胺金与半胱氨酸连接，4) 砷-金至四半胱氨酸标签，和5)金靶向金结合序列。 然后，这些标记将用于确定几个重要蛋白质复合物中的亚基位置： DNA 修复复合物由子复合物组成：人 Ku70/Ku80/DNA-PK 复合物、人连接酶 IV/Xrcc4 复合物、人 RAD50/Mre11/Nbs1 复合物和人类染色质重塑复合物 ACF。金色标签还将用于更好地对齐复合物，以改善使用冷冻电镜从锥形倾斜对进行的重建。